Estrogen receptors from the cytosol of calf uterus will be purified in large-scale by affinity chromatography using 17-beta-estradiol117- hemisuccinate coupled to agarose derivatives containing macromolecular spacers (branched copolymers of polylysylalanine). Transformed (4.5S) as well as native (5.3S) receptor proteins (and later the nuclear form of the receptor) will be purified. The physicochemical properties (subunit composition, molecular weight, amino acid composition, etc.) of the different forms of the purified receptor will be studied, and some important properties of the hormone-receptor interaction (stoichiometry, structural specificity, dissociation constants) will be determined. The receptor will be examined for possible enzymic activities. The receptor will be labeled (I125) to study directly its interaction with nuclei and separated nuclear components, and to examine the role of estrogens in this process. Receptor-agarose derivatives will be prepared to try to purify the specific nuclear-acceptor structures for the cytosol receptor. Specific antibodies against the purified receptors will be prepared and purified, and these will be used to a) prepare affinity columns which may be useful in purifying the receptors, b) develop a radioimmunoassay for quantitation of receptors in tissue extracts, c) study the in vivo effects of these antibodies on administered estrogens and on reproductive physiology, d) examine possible cross-reactivity with other steroid hormone-receptor interactions, and e) examine nature of receptors (and develop diagnostic assay methods) in breast cancers.